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Introduction

The intestinal mucosa, from duodenum to rectum, represents the largest physiological interface separating the environment from the systemic compartment [1]–[3]. Indeed, the mucosal surface area represents the most significant anatomic portal for systemic exposure to environmental toxins and microorganisms [4]. These exposures are prevented by the epithelial barrier, in part, comprising a network of junctional complexes that anneal membranes of neighboring enterocytes and thereby restrict paracellular macromolecular exchange between mucosal and systemic compartments [3]. While the molecular components of tight junctions have been enumerated, signaling mechanisms regulating dynamic programming of their steady-state concentrations, assembly, and deployment remain incompletely defined [1]–[3], [5]. In that context, signaling by v akt murine thymoma viral oncogene homolog (AKT) plays a central role in regulating barrier dynamics [6]–[9]. Importantly, there is an emerging recognition of the contribution of barrier dysfunction to local and systemic diseases, including inflammatory bowel disease, diabetes, multiple sclerosis, food allergy, asthma, systemic genotoxicity and malignancies [1]–[3], [10], [11].

Coordinated regulation of homeostatic processes maintaining the intestinal epithelium by GUCY2C is mediated by a molecular mechanism in which cGMP suppresses AKT phosphorylation and activation [15]. In turn, these homeostatic processes regulating epithelial integrity are essential components of molecular programs supporting mucosal barrier function [1]–[3], [10]. In the context of the established role of AKT in dynamic modulation of junctional complexes [6]–[9], we examined the impact of GUCY2C signaling on intestinal barrier integrity through AKT-dependent regulation of junctional complex components.

Materials and Methods

Animal models

Rosa-STOPflox-Guca2a-vil-Cre-ERT2 mice and corresponding genotype controls lacking Rosa-STOPflox-Guca2a insert were generated by crossbreeding vil-Cre-ERT2 (provided by Robine S., Institut Curie-CNRS, France) with Rosa-STOPflox-Guca2a transgenic mice (Thomas Jefferson University) (see Supplementary Information). C57BL6 mice used in oral ST supplementation studies were purchased from NIH (NCI-Frederick). Gucy2c−/−, Akt1−/−, Akt2−/− and corresponding wild type littermate mice were bred, maintained, genotyped, and functionally characterized as described in accordance with the Thomas Jefferson University Animal Care and Use guidelines. This study was approved by The Institutional Animal Care and Use Committee of Thomas Jefferson University under approved animal protocol 315O, and 315K [15].

DSS colitis

8 weeks old male or female mice were treated with 3 or 3.5% (w/v) DSS (Sigma-Aldrich) in their drinking water for 7 d followed by normal access to water (see Materials and Methods S1). Colitis severity was evaluated by body weight, mortality, and colon length, as well as histology scores. Colon length is a morphometric measure of the degree of colonic inflammation and correlates with pathologic changes related to colitis severity [20].

Histopathology scores

Colon tissues were fixed in 10% buffered formalin, processed and embedded in paraffin. Five µm tissue sections were stained with hematoxylin-eosin (H&E). Seven sections in each mouse were evaluated by two independent investigators blinded to genotype or experimental condition. Each section was scored using the sum of a 0–4 scale in both epithelial and mesenchymal compartments based on established semiquantitative criteria (see Table S1) [21].

Barrier integrity

Littermate mice were gavaged with 200 µL of FITC-dextran (75 mg/mL; 4 kD; Sigma-Aldrich) after an overnight fast. Hemolysis-free serum was analyzed for concentration of FITC-dextran by fluorimetry 90 min after gavage. Permeability of 4 kD FITC-dextran across Caco2 (ATCC) monolayers was assessed using the 24-mutiwell insert system (BD Biosciences).

DNA oxidation

DNA was extracted from leukocytes in Gucy2c+/+ and Gucy2c−/− mice, and from hepatocytes in mice treated with ST or control peptide followed by DSS administration, and stored at −20°C. DNA oxidation was estimated by quantifying 8-hydroxydeoxyguanosine employing the OxiSelect™ Oxidative DNA Damage ELISA Kit (Cell Biolabs, Inc., San Diego, CA).

Immunofluorescence

Distal colons were collected processed and embedded in paraffin blocks and then cut into 5 µM slides as previous described [15]. Tight junctions were stained by anti-claudin 4 antibody (Invitrogen) and epithelial cells were outlined by anti-β-catenin antibody (Santa Cruz Biotechnology). Nuclei were counterstained by DAPI.

Microarray analyses

Microarray analyses, using the Affymetrix Mouse 430 2.0 3′-IVT platform, were performed on RNA extracted from intestine of Gucy2c+/+ and Gucy2c−/− littermates (n=4 each) [15]. All data is MIAME compliant and that the raw data has been deposited in GEO, GeneExpressionOmnibus: a public functional genomics data repository supporting MIAME compliant submission, http://www.ncbi.nlm.nih.gov/geo/.

GUCY2C modulates homeostatic processes by opposing AKT1 phosphorylation and downstream signaling in intestine and colorectal cancer [15]. In the context of established AKT modulation of junctional complexes [6]–[9], steady-state levels of the tight junction proteins occludin and claudin 4, but not claudin 2 or JAM-A, were restored to those in wild type epithelial cells by normalizing phospho-AKT1 levels in Gucy2c−/−Akt1+/− mice (Fig. 6A). Restoration of occludin and claudin 4 was associated with reconstitution of intestinal barrier integrity characteristic of wild type mice by reducing AKT1, but not AKT2, expression in Gucy2c−/− mice (Fig. 6B).

Normalizing phospho-AKT1 levels in Gucy2c−/−Akt1+/− mice mimics the effect of GUCY2C on AKT1 in Caco2 human intestinal epithelial cells, an established in vitro model of the intestinal barrier [23]. Stimulating GUCY2C with ST reduced phospho-AKT1, an effect recapitulated by the downstream GUCY2C effector, cGMP (Fig. 6C). ST enhanced the barrier properties of these cells, decreasing the permeability of Caco2 monolayers (Fig. 6D), reproducing the in vivo effect of ST on intestinal permeability (Fig. 4A). The effect of ST on Caco2 permeability was mimicked by reducing AKT1 expression using a target-specific siRNA, recapitulating the effects of genetic knockdown of AKT1 in Gucy2c−/−Akt1+/− mice (Fig. 4B). Conversely, the effect of ST on Caco2 permeability was eliminated by a constitutively active AKT1 mutant, MyrAKT1 (Fig. 6D). Moreover, reducing the expression of AKT1 in Caco2 cells using a target-specific shRNA reproduced the effects of GUCY2C signaling on steady-state levels of occludin and claudin 4, but not claudin 2 or JAM-A (Fig. 6E), precisely recapitulating the effects of GUCY2C and AKT1 on junctional proteins in Gucy2c−/−Akt1+/− mice (Fig. 6A).

Discussion

Disruption of intestinal barrier integrity has profound pathophysiological consequences and there is an emerging recognition of the contribution of the “leaky gut” and intestinal hyperpermeability to human disease [1]–[3], [10], [24]. In part, intestinal barrier integrity reflects dynamic modulation of tight junction complexes through regulation of component proteins, including intracellular steady-state concentrations and their assembly and membrane deployment [2], [3]. These complexes are rate-limiting components in transepithelial transport and primary determinants of mucosal permeability [2], [3], [10]. Acquired or inherited impairment of this barrier is at least necessary, if not sufficient, for abnormal subepithelial exposure to lumenal antigens that provoke immune responses underlying inflammatory bowel disease [1], [2]. Beyond these local effects, epithelial barrier dysfunction leading to cross-compartmental exposure to environmental antigens may be one necessary precondition for the development of systemic multi-organ autoimmune diseases including type I diabetes, multiple sclerosis, asthma, food allergy, hypersensitivity and cancer [1]–[3], [10], [24]. These considerations underscore an unmet clinical need for novel therapeutic approaches that defend intestinal barrier integrity to prevent systemic disease.

While GUCY2C was originally identified as the intestinal receptor for the diarrheagenic bacterial STs, its role in modulating physiological fluid and electrolyte secretion emerged following the discovery of the endogenous paracrine hormones GUCA2A and GUCA2B [25]. Beyond fluid and electrolyte balance, recent studies have revealed a role for GUCY2C in regulating homeostatic programs that shape the dynamic crypt-villus axis. GUCY2C regulates key cell cycle circuits, restricting cell division as epithelial cells transition from the proliferating crypt to the differentiated villus compartment [13]–[16]. Also, GUCY2C drives differentiation along the secretory lineage, amplifying the production of goblet, Paneth and enteroendocrine cells [13]. Further, GUCY2C signaling shifts metabolic programming from glycolytic to mitochondrial oxidative metabolism as cells transition from proliferating to differentiated compartments [15]. Moreover, GUCY2C modulates DNA damage sensing and repair, protecting chromosomal integrity [13], [14]. Disruption of these homeostatic processes, reflecting universal early loss of expression of GUCA2A and GUCA2B which silences GUCY2C, contributes to hyperproliferation and chromosomal instability underlying intestinal tumorigenesis in animals and humans [16]–[19].

The present studies extend this homeostatic role of GUCY2C beyond proliferation, differentiation, migration, DNA damage sensing and repair, and metabolism to maintenance of macromolecular permeability and the intestinal barrier. Elimination of GUCY2C signaling produced basal macromolecular hyperpermeability and potentiated barrier disruption resulting in colitis induced by DSS [26]. Conversely, genetic or pharmacological induction of GUCY2C signaling reduced basal permeability and DSS-induced hyperpermeability and inflammation. Results in mouse models were recapitulated in monolayers of human intestinal epithelial cells in vitro, in the absence of other confounding (patho)physiological mechanisms like inflammation, demonstrating a primary mechanistic role for GUCY2C in regulating the epithelial barrier and macromolecular permeability. Moreover, protection of the epithelial barrier by GUCY2C in vivo and in vitro was associated with alterations in steady state concentrations of tight junction proteins, including occludin and claudin 4. Regulation of steady-state concentrations of these components are one established mechanism modulating the dynamic assembly and deployment of tight junction complexes, barrier integrity and macromolecular permeability [1]–[3], [10], [27].

Integration of crypt-villus homeostatic programs mediated by GUCY2C is coordinated through AKT1 [15]. Ligand activation of GUCY2C and accumulation of cGMP produces dephosphorylation of AKT1, modulating downstream signaling circuits regulating proliferation and metabolism [15]. In the present study, homeostatic regulation of epithelial barrier function and permeability by GUCY2C was mediated by AKT1. In that context, specifically reducing AKT1, but not AKT2, blocked alterations in occludin and claudin 4, but not JAM-A and claudin 2, and macromolecular hyperpermeability induced by eliminating GUCY2C signaling in mice. Further, in monolayers of human intestinal epithelial cells in vitro, eliminating AKT1 expression induced steady state levels of occludin and claudin 4, but not JAM-A and claudin 2, and barrier function to levels achieved by ST activation of GUCY2C. Moreover, a constitutively activated form of AKT1 produced resistance to, while eliminating AKT1 expression from these cells mimicked, the effects of GUCY2C activation on epithelial cell permeability. Together, these results demonstrate a key mechanistic role for suppression of AKT1 signaling in the regulation of epithelial cell barrier function by GUCY2C. These results reinforce the established role of AKT in regulating the expression of junctional proteins, disrupting epithelial cell membrane complexes, and inducing barrier dysfunction [6]–[9]. Further, they identify occludin and claudin 4, but not JAM-A and claudin 2, as specific downstream targets of AKT1 contributing to the regulation of epithelial barrier permeability in vitro and in vivo. They underscore the integrated homeostatic role of GUCY2C signaling, centrally coordinated through AKT, in regulating intestinal epithelial structure and function [12]–[15].

There is an established mechanistic relationship between disruption of the intestinal epithelial barrier, inflammation resulting from systemic immune exposure to normally compartmentalized lumenal antigens, and the generation of reactive oxygen species producing DNA damage and genotoxic stress [28]. In turn, reactive oxygen species producing DNA damage comprise one mechanism contributing to tumorigenesis generally, and to the evolution of colon cancer in the context of inflammatory bowel disease, specifically [11], [28]. Importantly, there is emerging recognition that this oxidative stress extends beyond the primary site of barrier disruption and inflammation, and is disseminated systemically [11]. Indeed, genetic or chemical disruption of the intestinal barrier associated with inflammation produces systemic DNA damage and genotoxic stress, including in circulating leukocytes [11]. Here, barrier disruption and hyperpermeability produced by silencing GUCY2C was associated with elevated levels of oxidative DNA damage in circulating leukocytes. Conversely, defending barrier integrity and restricting permeability by activating GUCY2C with oral ST decreased systemic genotoxicity in hepatocytes produced by DSS. Further, systemic genotoxicity produced by barrier disruption reflecting GUCY2C silencing was associated with increased spontaneous and carcinogen-induced extra-intestinal tumorigenesis, specifically in lung, liver and lymph nodes. In the context of the role of GUCY2C as a tissue-specific tumor suppressing receptor [12], the present observations expand that function beyond transformation in the colorectum [29], to neoplasia in lymph nodes, liver, and lung through maintenance of epithelial barrier integrity. Moreover, it is tempting to speculate that loss of paracrine hormone expression and silencing of GUCY2C in inflammatory bowel disease [30] produces systemic genotoxicity which contributes to the pattern of extra-intestinal cancer in these patients specifically in lymph nodes, liver, and lung [31], [32].

The present study further clarifies the evolving role of the GUCY2C paracrine hormone system in modulating the intestinal barrier. A role for GUCY2C in regulating epithelial barrier function was recently demonstrated, and the present work confirms that phenomenon [33]. Those studies revealed that silencing GUCY2C disrupted the barrier and induced inflammation, associated with production of interferon gamma and IL-12 [33]. In turn, those events were associated with increased expression of epithelial cell myosin light chain kinase, an established regulator of tight junction structure and function, and suppression of JAM-A and claudin 2 expression. These phenomena were presumed to be the molecular mechanisms mediating GUCY2C-dependent barrier disruption. However, canonical regulation of myosin light chain phosphorylation by cGMP is mediated by myosin light chain phosphatase, rather than myosin light chain kinase [34]. Moreover, these observations are complicated by the contribution of inflammatory cytokines, including interferon gamma, to regulation of expression of myosin light chain kinase in intestinal epithelial cells in vivo and in vitro[2], [3]. Thus, the contribution of these components may not reflect the primary mechanism underlying GUCY2C regulation of barrier integrity but, rather, an epiphenomenon related to the associated inflammation [33]. In contrast, the present study demonstrates a mechanistic role for GUCY2C as a primary modulator of the intestinal epithelial barrier, mediated by AKT1, occludin and claudin 4, but not JAM-A and claudin 2, in mouse models in vivo, and in human intestinal cell monolayers in vitro in the absence of confounding by inflammation.

Paradoxically, subsequent work from the same group suggested that eliminating GUCY2C signaling protects the intestinal mucosa from inflammatory injury [35]. In that study, barrier disruption by DSS induced greater inflammation, tissue damage, and mortality in Gucy2+/+, compared to Gucy2c−/−, mice [35]. These results stand in striking contradistinction to earlier reports by this group [33]. Indeed, while baseline inflammation was elevated in Gucy2c−/− mice previously [33], here these mice exhibited reduced levels of inflammatory cytokines including interferon gamma [35]. Similarly, while barrier disruption produced exaggerated responses in Gucy2c−/− mice previously [33], here barrier disruption produced disease of greater severity in Gucy2c+/+ mice [35]. Beyond the inconsistencies in these observations from the same group, they stand in striking contrast to the results presented herein, demonstrating that GUCY2C signaling defends mucosal integrity against barrier disruption and colitis produced by DSS. The importance of these inconsistencies can best be appreciated by their respective therapeutic implications. On the one hand, if cGMP signaling exacerbates barrier disruption resulting in inflammation, then GUCY2C antagonists could be therapeutic in inflammatory bowel disease. Conversely, if cGMP signaling defends the barrier and prevents ensuing inflammation, then GUCY2C agonists could be a significant addition to the therapeutic armamentarium against inflammatory bowel disease.

While the source remains unclear, one possibility for these and past inconsistencies [14], [36] is the animal models used for genetic comparisons [37]. Here, all comparisons between Gucy2c−/− and Gucy2c+/+ mice were performed with matched colony mates. This experimental design reflects our observation that wild type mice obtained commercially exhibit increased susceptibility to DSS compared to wild type mice from our colony (See Figure S1). If earlier studies [35], [36] compared the behavior of Gucy2c−/− or Guca2a−/− mice to commercially-available wild type mice, inconsistent results could reflect differences in genetic or environmental characteristics beyond the specific genes targeted in the models. The present study demonstrates the role of the GUCY2C paracrine axis in barrier regulation using multiple genetic models that either eliminate receptor circuits or constitutively express paracrine ligands, with appropriate intra-colony controls, complemented by studies of oral GUCY2C ligand efficacy conducted in wild type mice from an independent colony. This evidence strongly supports a role for the GUCY2C paracrine axis in maintaining barrier integrity, and the therapeutic value of GUCY2C ligands in preventing and treating barrier disruption and inflammatory bowel disease.

Beyond proliferation, metabolism, and chromosomal integrity, the present studies reveal an additional dimension of AKT1-dependent epithelial homeostatic regulation by GUCY2C. Indeed, it is tempting to speculate that one essential function of GUCY2C signaling is maintenance of intestinal barrier integrity. In that context, regulation of epithelial tight junctions contributing to macromolecular permeability described here can be added to other established GUCY2C functions, including fluid and electrolyte secretion accelerating lumenal clearance; differentiation of Paneth cells producing antimicrobial peptides; and differentiation of goblet cells producing intestinal mucus that contribute to the separation of systemic and environmental compartments across the mucosal interface [13]. The pathophysiological significance of this role in intestinal barrier protection is underscored by the impact of GUCY2C signaling on colitis and systemic genotoxicity and tumorigenesis. In turn, these considerations highlight the translational opportunities for GUCY2C ligands in the prevention and treatment of inflammatory bowel disease and extra-intestinal malignancies. This therapeutic potential is underscored by the recent advancement of the oral GUCY2C ligand linaclotide for FDA approval for the treatment of constipation-type irritable bowel syndrome [38].

Supporting Information

Figure S1

Environmental effects on DSS-induced colitis. 8 week old C57JBL6 mice purchased from NCI (black circles) and in-house C57JBL6 mice generated from Gucy2c+/− colony (blue circles) were fed with 3% DSS for 7 d followed by regular drinking water. Body weight (n≥11) was measured as an indicator for severity of colitis. Values are given as percentage of body weight on day 0±SEM. *, p<0.05.

Table S1

Histopathology score table. Semiquantitative histopathological scoring system based on the criteria presented in the table was used by two independent investigators blinded to genotypes or experimental condition to assess severity of colitis. Scores from epithelium and mesenchyme were summed to determine the degree of inflammation for each section. Mean scores of at least seven sections per animal were presented in figures.

Data S1

Footnotes

Competing Interests: The authors have read the journal's policy and have the following conflicts: This study was partly funded by Targeted Diagnostic and Therapeutics Inc. Dr. Waldman is the Chair (uncompensated) of the Scientific Advisory Board of Targeted Diagnostics and Therapeutics, Inc., which has a license to commercialize inventions related to this work. Dr. Waldman is the Samuel M.V. Hamilton Endowed Professor. There are no patents, products in development or marketed products to declare. This does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials, as detailed online in the guide for authors.

Funding: These studies were supported by grants from the National Institutes of Health (NIH) (CA146033), the Pennsylvania Department of Health, and Targeted Diagnostic and Therapeutics Inc. The Pennsylvania Department of Health specifically disclaims responsibility for any analyses, interpretations or conclusions. Dr. Garcia and Dr. Li were supported by NIH 5T32 GM08562. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.